GSM7179034: A549-MSTRG-4401-1_KO_S24; Homo sapiens; RNA-Seq
Genome editing using CRISPR/Cas9 was used to knockout the lncRNAs' DNA binding domains (DBDs). Before and after the knockout, RNA-sequencing was performed to examine gene expression (performed by HaploX Genomics Center, Shenzhen, China, https://www.haplox.cn/pc/home). Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The quality and purity of RNA were examined by a NanoDrop™ One/OneC spectrophotometer (Thermo Scientific, Waltham, MA, USA) and Life Invitrogen Qubit RNA BR (Broad-Range) Assay Kit. RNA integrity was analyzed using Agilent 4200 TapeStation system (Agilent, Santa Clara, CA, USA). 1 μg of total RNA per sample was prepared for library preparation. Firstly, Ribo-Zero rRNA Removal Reagent (H/M/R) (Illumina, USA) was used to remove rRNA. Then, Magmetic RNA Beads (Vazyme, China) was used to purify remaining RNA. Finally, library was constructed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) and VAHTS DNA Clean Beads (Vazyme, China) and Q5 Hot Start High-Fidelity 2X Master Mix and primer mix (NEB, USA) was used to amplify library.The concentration of constructed library was measured by Qubit®3.0 Fluorometer and Library molar concentration was measured by QuantStudio 3 qPCR, and Agilent 4200 was used to examine the distribution of segments in library. Finally, RNA-Seq was performed on an Illumina NovaSeq6000 platform according to the manufacturer's instructions